Cloned leptospira outer membrane protein

ABSTRACT

An antigenic preparation is provided which contains a 31 Kd outer membrane protein from Leptospira which can be used immunologically as a vaccine for leptospirosis caused by this organism.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates generally to an antigenic preparation andspecifically to a Leptospira outer membrane protein (OmpL1) which isused to induce a protective immune response in animals. Such a proteincan be used immunologically as a vaccine for leptospirosis caused bythis organism. Alternatively, diagnosis of leptospirosis can beperformed by detecting the presence of the protein, antibody to theprotein, or polynucleotide which encodes the protein.

[0003] 2. Description of Related Art

[0004] Leptospirosis is a widespread zoonotic disease caused bypathogenic strains of Leptospira which are capable of infecting mostmammalian species. At present, there are six pathogenic species andthree nonpathogenic species within the genus Leptospira. Infectionoccurs either through direct contact with an infected animal or indirectcontact with contaminated soil or water. In livestock, the diseasecauses economic losses due to abortion, stillbirth, infertility,decreased milk production, and death.

[0005] Efforts to control leptospirosis have been hampered becausevirulent leptospires have the capacity for both long-term survival inthe environment as well as persistent infection and shedding by wildlifeand livestock. Currently available leptospiral vaccines produceshort-term immunity and do not provide cross-protection against many ofthe 170 serovars of pathogenic Leptospira (Thiermann, et al.,J.Am.Vet.Med.Assoc. 184:722, 1984). These vaccines consist ofinactivated whole organisms or outer envelope preparations which produceseroreactivity as determined by microscopic agglutination of intactorganisms. The nature of the protective immunogens in these vaccinepreparations has not been conclusively elucidated, although severallines of evidence suggest that lipopolysaccharide-like substance (LLS)may confer a degree of protection.

[0006] The pathogenesis of leptospirosis is very similar to that ofother spirochetal diseases, including syphilis (caused by Treponemapallidum) and Lyme borreliosis (caused by Borrelia burgdorferi). Bothsyphilis and Lyme borreliosis are characterized by widespreaddissemination early in the course of disease, including invasion of thecentral nervous system. Leptospira share this ability with otherpathogenic spirochetes such that meningitis is a common manifestation ofleptospirosis. Another feature of spirochetal infections is the abilityto persist chronically in the host, as manifested in cases of tertiarysyphilis and chronic Lyme arthritis.

[0007] In attempting to identify leptospiral outer membrane proteins(OMPs), previous research was unsuccessful due to such problems as: 1)the techniques used to identify surface-exposed proteins probablyinvolved damage to the fragile leptospiral outer membrane resulting inexposure of subsurface structures; 2) putative surface-exposed proteinsthat were identified included a 35-36 kD doublet corresponding toLeptospira endoflagella (Kelson, et al., J. Med. Microbiol. 26:47,1988), which are subsurface structures in spirochetes; and 3) use of SDSwhich nonselectively solubilizes proteins irrespective of their nativecellular location.

[0008] Nunes-Edwards, et al. (Infect. Immun. 48:492, 1985) introducedthe use of radioimmunoprecipitation and cell fractionation schemes basedon the use of SDS in an effort to identify leptospiral OMPs. Theleptospires used in their radioimmunoprecipitation procedure weresubjected to high speed centrifugation (20,000×g) prior to the additionof antibody. Such high centrifugal forces cause mechanical disruption ofthe leptospiral outer membrane. Niikura, et al (Zbl. Bakt. Hyg. A.266:453, 1987) immunoprecipitated SDS-solubilized extracts of virulentand avirulent strains of L. interrogans serovar copenhageni that hadbeen labeled by lactoperoxidase-catalyzed surface radioiodination. Sinceboth of these studies precipitated a 35-36 kD doublet consistent withleptospiral endoflagella, there was a concern as to whether the otherproteins identified might also have a subsurface rather than a surfacelocation.

[0009] Jost, et al. (J. Med. Microbiol. 27:143) characterized, amonoclonal antibody with specificity for a 35 kD proteinase K sensitiveantigen which was present in a leptospiral outer envelope preparation.However, to demonstrate binding of the monoclonal antibody byimmunoelectron microscopy, the leptospiral outer membrane had to bedisrupted. Doherty, et al. (J. Med. Microbiol. 28:143) cloned twoleptospiral proteins represented in an SDS-generated outer membranepreparation of L. interrogans, but did not provide corroboratingevidence that these proteins are either constituents of the outermembrane or are surface-exposed.

[0010] Unsuccessful research on the identification of Leptospira and T.pallidum OMPs has shown the importance of taking into accountspirochetal outer membrane fragility and the lack of outer membraneselectivity of ionic detergents such as sodium dodecyl sulfate (SDS)(Cunningham, et al, J.Bacteriol. 170:5789, 1988; Penn, et al., J. Gen.Microbiol. 131:2349, 1985; Stamm, et al., Infect. Immun. 55:2255, 1987).Outer membrane proteins are of great importance because they play a keyrole in bacterial pathogenesis. The identification of outer membraneproteins involved in Leptospira pathogenesis is significant tounderstanding not only leptospiral outer membrane proteins and theirinvolvement in pathogenesis, but also to understanding other spirochetalouter membrane proteins and their role in pathogenesis.

SUMMARY OF THE INVENTION

[0011] The present invention is based on the identification of OmpL1 asa major leptospiral outer membrane protein which is associated withpathogenic strains of Leptospira. Due to spirochetal outer membranefragility and the fact that outer membrane proteins are present in smallamounts, there have been no definitive reports of membrane spanningspirochetal outer membrane proteins until the present invention. Theinvention describes a 31 kD outer membrane protein from Leptospira andthe gene encoding the protein. This gene is present in all species ofpathogenic Leptospira tested and is absent in all nonpathogenicLeptospira tested. The deduced amino acid sequence has a typical leaderpeptidase I cleavage site, implying export beyond the inner membrane.The 31 kD protein has been designated OmpL1 for outer membrane proteinof Leptospira. This immunogenic polypeptide is useful for inducing animmune response to pathogenic Leptospira as well as providing adiagnostic target for leptospirosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows the DNA sequence and deduced amino acid sequence ofOmpL1.

[0013]FIG. 2 shows a comparison between Nmp1 (an outer membrane proteinporin of Neisseria meningitidis and OmpL1 amino acid sequences.

[0014]FIG. 3A shows a Southern blot using ompL1 as a probe to detect thegene in pathogenic and non-pathogenic Leptospira (medium stringency).

[0015]FIG. 3B shows a Southern blot using ompL1 as a probe to detect thegene in pathogenic and non-pathogenic Leptospira (high stringency).

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides an isolated immunogenicpolypeptide from an outer membrane protein of a pathogenic Leptospiraspecies. Also included is a polynucleotide sequence which encodes thepolypeptide. The outer membrane protein is a 31 kD protein originallyisolated from Leptospira alstoni which has been termed OmpL1 and is apathogen-associated exported protein of Leptospira. This immunogenicpolypeptide is useful in a pharmaceutical composition for inducing animmune response to pathogenic Leptospira.

[0017] The invention includes a method of producing the polypeptideportion of an outer membrane protein of Leptospira using recombinant DNAtechniques. The gene for the L. alstoni OmpL1 outer membrane protein wascloned into a plasmid vector which was then used to transform E. coli.When the OmpL1 gene was expressed in E. coli, the polypeptide producedhad a molecular weight of approximately 31 kD as determined bySDS-polyacrylamide gel electrophoresis. Reactivity to the 31 kD proteinwas demonstrated with antisera to pathogenic strains of Leptospiraincluding L. interrogans serovars icterohae-morrhagiae, pomona andbratislava, L. aistoni, serovars grippotyphosa and fortbragg, L.santarosai, serovars bakeri and canalzonae, and L. weilii, serovarcelledoni. This indicates that OmpL1 is not only expressed, but alsoantigenically conserved among pathogenic Leptospira regardless ofspecies and, therefore, this polypeptide is an excellent vaccinecandidate as well as a marker antigen for diagnosis of leptospirosis.

[0018] Extraction of proteins from whole cells of L. alstoni usingnonionic detergent Triton X-114 (TX-114), resulted in the solubilizationof a number of proteins, including a detergent phase protein of 31 kD(OmpL1). Surface immunoprecipitation using antiserum raised to whole L.alstoni, was used to generate a fraction which was subjected toSDS-polyacrylamide gel electrophoresis. The electrophoresed fraction wasthen transferred to a sequencing membrane and an N-terminal sequence of14 amino acids of the 31 kD protein was determined. Based upon theN-terminal amino acid sequence, two degenerate oligonucleotide probeswere synthesized. An L. alstoni genomic DNA library was probed with theoligonucleotides and a 2.5 kb insert was identified as containing thecoding sequence for 31 kD OmpL1.

[0019] Sequence analysis showed that the OmpL1 structural gene consistsof 960 bases encoding a protein of 320 amino acids. As expected forproteins to be exported beyond the inner membrane, the derived aminoacid sequence begins with a 24-residue signal peptide. The OmpL1sequence contains ten stretches of amphipathic beta-sheet structure,consistent with outer membrane protein transmembrane segments. Southernhybridization studies showed that there is a strong correlation betweenLeptospira pathogenicity and the presence of the OmpL1 gene. A singlecopy of the OmpL1 gene was present in all strains of pathogenicLeptospira tested, and absent in all nonpathogenic strains of Leptospiratested.

[0020] The bacterial genes for the OmpL1 outer membrane protein can bederived from any strain of pathogenic Leptospira. Preferably the proteinis from Leptospira alstoni, serovar grippotyphosa.

[0021] The invention provides polynucleotides encoding the LeptospiraOmpL1 protein. These polynucleotides include DNA and RNA sequences whichencode the protein. It is understood that all polynucleotides, encodingall or a portion of OmpL1 are also included herein, so long as theyexhibit a function of OmpL1, such as the ability to induce or bindantibody. Such polynucleotides include both naturally occurring andintentionally manipulated, for example, mutagenized polynucleotides. DNAsequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic libraries to detect shared nucleotidesequences and 2) antibody screening of expression libraries to detectshared structural features.

[0022] Hybridization procedures are useful for the screening ofrecombinant clones by using labeled mixed synthetic oligonucleotideprobes where each probe is potentially the complete complement of aspecific DNA sequence in the hybridization sample which includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. By using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific DNA clone by the hybridization of the target DNA to that singleprobe in the mixture which is its complete complement (Wallace, et al.,Nucleic Acid Research, 9:879, 1981).

[0023] Alternatively, an expression library can be screened indirectlyfor OmpL1 peptides having at least one epitope using antibodies toOmpL1. Such antibodies can be either polyclonally or monoclonallyderived and used to detect expression product indicative of the presenceof OmpL1 DNA. Generally, a lambda gt11 library is constructed andscreened immunologically according to the method of Huynh, et al. (inDNA Cloning:A Practical Approach, D. M. Glover, ed., 1:49, 1985).

[0024] The development of specific DNA sequences encoding OmpL1 can alsobe obtained by: (1) isolation of a double-stranded DNA sequence from thegenomic DNA, and (2) chemical manufacture of a DNA sequence to providethe necessary codons for the polypeptide of interest. DNA sequencesencoding OmpL1 can be expressed in vitro by DNA transfer into a suitablehost cell. “Recombinant host cells” or “host cells” are cells in which avector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that not allprogeny are identical to the parental cell since there may be mutationsthat occur at replication. However, such progeny are included when theterms above are used.

[0025] The term “host cell” as used in the present invention is meant toinclude not only prokaryotes, but also, such eukaryotes as yeasts,filamentous fungi, as well as plant and animal cells. The term“prokaryote” is meant to include all bacteria which can be transformedwith the gene for the expression of the OmpL1 outer membrane protein ofLeptospira. Prokaryotic hosts may include Gram negative as well as Grampositive bacteria, such as E. coli, S. typhimurium, and Bacillussubtilis.

[0026] A recombinant DNA molecule coding for the OmpL1 protein can beused to transform a host using any of the techniques commonly known tothose of ordinary skill in the art. Especially preferred is the use of aplasmid containing the OmpL1 coding sequence for purposes of prokaryotictransformation. Where the host is prokaryotic, such as E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method by procedures well known in the art. Alternatively,MgCl₂ or RbCl can be used. Transformation can also be performed afterforming a protoplast of the host cell.

[0027] In the present invention, the OmpL1 sequences may be insertedinto a recombinant expression vector. The term “recombinant expressionvector” refers to a plasmid, virus or other vehicle known in the artthat has been manipulated by insertion or incorporation of OmpL1 geneticsequences. Such expression vectors contain a promotor sequence whichfacilitates the efficient transcription of the inserted genetic sequencein the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. The transformedprokaryotic hosts can be cultured according to means known in the art toachieve optimal cell growth. Various shuttle vectors for the expressionof foreign genes in yeast have been reported (Heinemann, et al., Nature,340:205, 1989; Rose, et al., Gene, 60:237, 1987). Biologicallyfunctional DNA vectors capable of expression and replication in a hostare known in the art. Such vectors are used to incorporate DNA sequencesof the invention.

[0028] Methods for preparing fused, operably linked genes and expressingthem in bacteria are known and are shown, for example, in U.S. Pat. No.4,366,246 which is incorporated herein by reference. The geneticconstructs and methods described therein can be utilized for expressionof Leptospira OmpL1 in prokaryotic hosts.

[0029] Examples of promoters which can be used in the invention are: recA, trp, lac, tac, and bacteriophage lambda p_(R) or p_(L). Examples ofplasmids which can be used in the invention are listed in Maniatis, etal.,(Molecular Cloning, Cold Spring Harbor Laboratories, 1982).

[0030] Antibodies provided in the present invention are immunoreactivewith OmpL1 protein. Antibody which consists essentially of pooledmonoclonal antibodies with different epitopic specificities, as well asdistinct monoclonal antibody preparations are provided. Monoclonalantibodies are made from antigen containing fragments of the protein bymethods well known in the art (Kohler, et al., Nature, 256:495, 1975;Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989). Theterm antibody, or immunoglobulin, as used in this invention includesintact molecules as well as fragments thereof, such as Fab and F(ab)₂,which are capable of binding an epitopic determinant on OmpL1.

[0031] Minor modifications of OmpL1 primary amino acid sequence mayresult in proteins which have substantially equivalent function comparedto the OmpL1 protein described herein. Such modifications may bedeliberate, as by site-directed mutagenesis, or may be spontaneous. Allproteins produced by these modifications are included herein as long asOmpL1 function exists.

[0032] Modifications of OmpL1 primary amino acid sequence also includeconservative variations. The term “conservative variation” as usedherein denotes the replacement of an amino acid residue by another,biologically similar residue. Examples of conservative variationsinclude the substitution of one hydrophobic residue such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of. arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like. The term “conservative variation” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

[0033] Isolation and purification of microbially expressed protein, onfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

[0034] The invention extends to any host modified according to themethods described, or modified by any other methods, commonly known tothose of ordinary skill in the art, such as, for example, by transfer ofgenetic material using a lysogenic phage, and which result in aprokaryote expressing the Leptospira gene for OmpL1 protein. Prokaryotestransformed with the Leptospira gene encoding the OmpL1 protein areparticularly useful for the production of polypeptides which can be usedfor the immunization of an animal.

[0035] In one embodiment, the invention provides a pharmaceuticalcomposition useful for inducing an immune response to pathogenicLeptospira in an animal comprising an immunologically effective amountof OmpL1 in a pharmaceutically acceptable carrier. The term“immunogenically effective amount,” as used in describing the invention,is meant to denote that amount of Leptospira antigen which is necessaryto induce in an animal the production of an immune response toLeptospira. The OmpL1 outer membrane protein of the invention isparticularly useful in sensitizing the immune system of an animal suchthat, as one result, an immune response is produced which amelioratesthe effect of Leptospira infection.

[0036] The OmpL1 outer membrane protein can be administered parenterallyby injection, rapid infusion, nasopharyngeal absorption, dermalabsorption, and orally. Pharmaceutically acceptable carrier preparationsfor parenteral administration include sterile or aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Carriers forocclusive dressings can be used to increase skin permeability andenhance antigen absorption. Liquid dosage forms for oral administrationmay generally comprise a liposome solution containing the, liquid dosageform. Suitable forms for suspending the liposomes include emulsions,suspensions, solutions, syrups, and elixirs containing inert diluentscommonly used in the art, such as purified water. Besides the inertdiluents, such compositions can also include adjuvants, wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

[0037] It is also possible for the antigenic preparations containing theOmpL1 protein of the invention to include an adjuvant. Adjuvants aresubstances that can be used to nonspecifically augment a specific immuneresponse. Normally, the adjuvant and the antigen are mixed prior topresentation to the immune system, or presented separately, but into thesame site of the animal being immunized. Adjuvants can be looselydivided into several groups based on their composition. These groupsinclude oil adjuvants (for example, Freund's Complete and Incomplete),mineral salts (for example, AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica,alum, Al(OH)₃, Ca₃(PO₄)₂, kaolin, and carbon), polynucleotides (forexample, poly IC and poly AU acids), and certain natural substances (forexample, wax D from Mycobacterium tuberculosis, as well as substancesfound in Corynebacterium parvum, Bordetella pertussis, and members ofthe genus Brucella).

[0038] In another embodiment, a method of inducing an immune response topathogenic Leptospira in animal is provided. Many different techniquesexist for the timing of the immunizations when a multiple immunizationregimen is utilized. It is possible to use the antigenic preparation ofthe invention more than once to increase the levels and diversity ofexpression of the immune response of the immunized animal. Typically, ifmultiple immunizations are given, they will be spaced two to four weeksapart. Subjects in which an immune response to Leptospira is desirableinclude swine, cattle and humans.

[0039] Generally, the dosage of OmpL1 protein administered to an animalwill vary depending on such factors as age, condition, sex and extent ofdisease, if any, and other variables which can be adjusted by one ofordinary skill in the art. The antigenic preparations of the inventioncan be administered as either single or multiple dosages and can varyfrom about 10 ug to about 1,000 ug for the Leptospira OmpL1 antigen perdose, more preferably from about 50 ug to about 700 ug OmpL1 antigen perdose, most preferably from about 50 ug to about 300 ug OmpL1 antigen perdose.

[0040] When used for immunotherapy, the monoclonal antibodies of theinvention may be unlabeled or labeled with a therapeutic agent. Theseagents can be coupled either directly or indirectly to the monoclonalantibodies of the invention. One example of indirect coupling is by useof a spacer moiety. These spacer moieties, in turn, can be eitherinsoluble or soluble (Diener, et al., Science, 231:148, 1986) and can beselected to enable drug release from the monoclonal antibody molecule atthe target site. Examples of therapeutic agents which can be coupled tothe monoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

[0041] The labeled or unlabeled monoclonal antibodies of the inventioncan also be used in combination with therapeutic agents such as thosedescribed above. Especially preferred are therapeutic combinationscomprising the monoclonal antibody of the invention and immunomodulatorsand other biological response modifiers.

[0042] When the monoclonal antibody of the invention is used incombination with various therapeutic agents, such as those describedherein, the administration of the monoclonal antibody and thetherapeutic agent usually occurs substantially contemporaneously. Theterm “substantially contemporaneously” means that the monoclonalantibody and the therapeutic agent are administered reasonably closetogether with respect to time. Usually, it is preferred to administerthe therapeutic agent before the monoclonal antibody. For example, thetherapeutic agent can be administered 1 to 6 days before the monoclonalantibody. The administration of the therapeutic agent can be daily, orat any other interval, depending upon such factors, for example, as thenature of the disorder, the condition of the patient and half-life ofthe agent.

[0043] The dosage ranges for the administration of monoclonal antibodiesof the invention are those large enough to produce the desired effect inwhich the onset symptoms of the leptospiral disease are ameliorated. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the subject and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any complication. Dosage can vary from about 0.1 mg/kg to about2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one ormore dose administrations daily, for one or several days. Generally,when the monoclonal antibodies of the invention are administeredconjugated with therapeutic agents, lower dosages, comparable to thoseused for in vivo diagnostic imaging, can be used.

[0044] The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells.

[0045] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

[0046] In a further embodiment, the invention provides a method ofdetecting a pathogenic Leptospira-associated disorder in a subjectcomprising contacting a cell component with a reagent which binds to thecell component. The cell component can be nucleic acid, such as DNA orRNA, or it can be protein. When the component is nucleic acid, thereagent is a nucleic acid probe or PCR primer. When the cell componentis protein, the reagent is an antibody probe. The probes are detectablylabeled, for example, with a radioisotope, a fluorescent compound, abioluminescent compound, a chemiluminescent compound, a metal chelatoror an enzyme. Those of ordinary skill in the art will know of othersuitable labels for binding to the antibody, or will be able toascertain such, using routine experimentation.

[0047] For purposes of the invention, an antibody or nucleic acid probespecific for OmpL1 may be used to detect the presence of OmpL1polypeptide (using antibody) or polynucleotide (using nucleic acidprobe) in biological fluids or tissues. Any specimen containing adetectable amount of OmpL1 antigen or polynucleotide can be used. Apreferred specimen of this invention is blood, urine, cerebrospinalfluid, or tissue of endothelial origin.

[0048] When the cell component is nucleic acid, it may be necessary toamplify the nucleic acid prior to binding with a Leptospira specificprobe. Preferably, polymerase chain reaction (PCR) is used, however,other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used.

[0049] Another technique which may also result in greater sensitivityconsists of coupling antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, pyridoxal, and fluorescein, which canreact with specific antihapten antibodies.

[0050] Alternatively, OmpL1 polypeptide can be used to detect antibodiesto OmpL1 polypeptide in a specimen. The OmpL1 of the invention isparticularly suited for use in immunoassays in which it can be utilizedin liquid phase or bound to a solid phase carrier. In addition, OmpL1used in these assays can be detectably labeled in various ways.

[0051] Examples of immunoassays which can utilize the OmpL1 of theinvention are competitive and noncompetitive immunoassays in either adirect or indirect format. Examples of such immunoassays are theradioimmunoassay (RIA), the sandwich (immunometric assay) and theWestern blot assay. Detection of antibodies which bind to the OmpL1 ofthe invention can be done utilizing immunoassays which run in either theforward, reverse, or simultaneous modes, including immunohistochemicalassays on physiological samples. The concentration of OmpL1 which isused will vary depending on the type of immunoassay and nature of thedetectable label which is used. However, regardless of the type ofimmunoassay which is used, the concentration of OmpL1 utilized can bereadily determined by one of ordinary skill in the art using routineexperimentation.

[0052] The OmpL1 of the invention can be bound to many differentcarriers and used to detect the presence of antibody specificallyreactive with the polypeptide. Examples of well-known carriers includeglass, polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amyloses, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The nature of thecarrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding OmpL1 or will be able to ascertain such, using routineexperimentation.

[0053] There are many different labels and methods of labeling known tothose of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, colloidal metals, fluorescent compounds, chemiluminescentcompounds, and bioluminescent compounds.

[0054] For purposes of the invention, the antibody which binds to OmpL1of the invention may be present in various biological fluids andtissues. Any sample containing a detectable amount of antibodies toOmpL1 can be used. Normally, a sample is a liquid such as urine, saliva,cerebrospinal fluid, blood, serum and the like, or a solid or semi-solidsuch as tissue, feces and the like.

[0055] The monoclonal antibodies of the invention, directed towardOmpL1, are also useful for the in vivo detection of antigen. Thedetectably labeled monoclonal antibody is given in a dose which isdiagnostically effective. The term “diagnostically effective” means thatthe amount of detectably labeled monoclonal antibody is administered insufficient quantity to enable detection of Leptospira OmpL1 antigen forwhich the monoclonal antibodies are specific.

[0056] The concentration of detectably labeled monoclonal antibody whichis administered should be sufficient such that the binding to thosecells, body fluid, or tissue having OmpL1 is detectable compared to thebackground. Further, it is desirable that the detectably labeledmonoclonal antibody be rapidly cleared from the circulatory system inorder to give the best target-to-background signal ratio.

[0057] As a rule, the dosage of detectably labeled monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sex,and extent of disease of the subject. The dosage of monoclonal antibodycan vary from about 0.001 mg/m² to about 500 mg/m², preferably 0.1 mg/m²to about 200 mg/m², most preferably about 0.1 mg/m² to about 10 mg/m².Such dosages may vary, for example, depending on whether multipleinjections are given, and other factors known to those of skill in theart.

[0058] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 key range, which may be readilydetected by conventional gamma cameras.

[0059] For in vivo diagnosis, radioisotopes may be bound toimmunoglobulin either directly or indirectly by using an intermediatefunctional group. Intermediate functional groups which often are used tobind radioisotopes which exist as metallic ions to immunoglobulins arethe bifunctional chelating agents such as diethylenetriaminepentaceticacid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to themonoclonal antibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²AS, ⁸⁹Zr, and ²⁰¹TI.

[0060] The monoclonal antibodies of the invention can also be labeledwith a paramagnetic isotope for purposes of in vivo diagnosis, as inmagnetic resonance imaging (MRI) or electron spin resonance (ESR). Ingeneral, any conventional method for visualizing diagnostic imaging canbe utilized. Usually gamma and positron emitting radioisotopes are usedfor camera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

[0061] The monoclonal antibodies of the invention can be used to monitorthe course of amelioration of Leptospira associated disorder. Thus, bymeasuring the increase or decrease of Leptospira OmpL1 polypeptide orantibodies to OmpL1 polypeptide present in various body fluids ortissues, it would be possible to determine whether a particulartherapeutic regiment aimed at ameliorating the disorder is effective.

[0062] The materials for use in the method of the invention are ideallysuited for the preparation of a kit. Such a kit may comprise a carriermeans being compartmentalized to receive in close confinement one ormore container means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise a OmpL1binding reagent, such as an antibody. A second container may furthercomprise OmpL1 polypeptide. The constituents may be present in liquid orlyophilized form, as desired.

[0063] The following examples are intended to illustrate but not limitthe invention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES

[0064] The following examples describe the identification of OmpL1 as animportant leptospiral OMP candidate. A comprehensive study of L. alstonisurface components is presented, including nonionic detergent extractionof the leptospiral outer membrane, surface immunoprecipitation, andfreeze-fracture electron microscopy. The method by which the ompL1 genewas cloned and sequenced is described. Sequence analysis and homologystudies are shown, further indicating that OmpL1 is an OMP. Southernhybridization and immunoblot studies show that OmpL1 is relevant to abroad range of pathogenic Leptospira, regardless of the species.

Example 1 Triton X-114 Nonionic Detergent Extraction of the OuterMembrane

[0065] The approaches used to characterize surface components weredesigned to address problems inherent in the known fragility of thespirochetal outer membrane (OM). The first approach involved the use ofthe nonionic detergent Triton X-114 (TX-114) which has the usefulcharacteristic of partitioning into hydrophobic and hydrophilic phasesupon warming. Membrane proteins are hydrophobic and typicallyfractionate into the hydrophobic phase, while soluble, non-membraneproteins fractionate into the hydrophilic phase. Several lines ofevidence suggested that 0.1% TX-114 selectively solubilized theleptospiral OM. TX-114 treatment reduced the leptospiral diameter withloss of discernable OM structure and resulted in essentially completerelease of LLS. The TX-114 soluble fraction was free of periplasmicflagella as determined by immunoblot probed with cross-reactiveantiserum specific for T. pallidum endoflagella. This finding representsthe first demonstration of epitopic conservation between endoflagella ofT. pallidum and Leptospira.

[0066] Extraction of L. alstoni using 0.1% TX-114 resulted in thesolubilization of a number of proteins varying in molecular weight fromgreater than 100 kDa to less than 14 kDa. Roughly 16 proteinspartitioned into the hydrophobic, detergent phase. Detergent phaseproteins of 35-, 39-, and 66-kDa appeared to be unique to the virulentstrain. By comparison, detergent phase proteins of 31-kDa (OmpL1) and38-kDa appeared to be expressed in greater amounts in the attenuatedstrain. The virulent and attenuated strains contained similar amounts ofthe major detergent phase proteins with molecular weights of 41- and44-kDa. The presence of LLS in leptospiral samples subjected toSDS-polyacrylamide gel electrophoresis was manifested by acharacteristic pattern of bands upon either periodate-silver staining orimmunoblotting. TX-114 studies demonstrated that leptospiral LLSpartitioned exclusively into the hydrophobic phase, confirming that LLSis a hydrophobic, outer membrane component. This result is alsoconsistent with the hydrophobic nature of the long-chain fatty acidcomponent of LLS.

Example 2 Surface Immunoprecipitation of Outer Membrane Conponents

[0067] The second approach used to identify surface components of L.alstoni was surface immunoprecipitation using antiserum raised to wholeorganisms. In order to avoid physical manipulation of the fragilespirochetes, a modification of the surface immunoprecipitation techniqueof Hansen, et al. (infect Immun. 31:950-953, 1981), was used whichinvolved addition of antiserum to a culture of intact, motile Leptospirain the log-phase of growth. The agglutinated Leptospira were then washedfree of unbound antibody using low speed centrifugation (2,000×g) inorder to avoid disruption of the outer membrane. The antigen-antibodycomplexes were solubilized in the Hansen solubilization buffer, andisolated using Staphylococcal protein A-Sepharose (CL-4B (Sigma)).Immunoblot of the immunoprecipitated material did not detect appreciableamount of the 35-36-kDa doublet endoflagellar protein, supporting theselectivity of the immunoprecipitation procedure for the identificationof leptospiral surface components.

[0068] The surface immunoprecipitation procedure provided informationcorroborating the surface location of the 31-kDa (OmpL1), 41-kDa, and44-kDa proteins. The 31-kDa (OmpL1) protein was present in significantlygreater amounts in culture-attenuated L. alstoni than in virulent L.alstoni. The 41-kDa and 44-kDa proteins were present in similar amountsin the two strains. Surface immunoprecipitation results providedevidence demonstrating that LLS epitopes of L. alstoni have surfaceexposure on living cells; earlier radioimmunoprecipitation studiesfocused entirely on the surface exposure of protein epitopes.

Example 3 Determination of Omp Content by Freeze-fracture ElectronMicroscopy

[0069] Suspensions containing approximately 4×10⁸ viable spirochetes(100% motility), as determined by enumeration of 20 fields by dark-fieldmicroscopy (at least 200 total organisms), were centrifuged at 30,000×gto pellet the organisms. The pellets were suspended in 2 ml of 2%glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). After 15 minof fixation, 1 ml of the suspension was transferred to each of two1.5-ml microfuge centrifuge tubes and the treponemes were pelleted bycentrifugation at 14,000×g. The pellets were suspended in 50 μl of 20%glycerol in 0.1 M sodium cacodylate buffer (pH 7.4). These manipulationswere performed at ambient temperature (22 to 24° C.). From thissuspension, 2-μl portions were placed on standard Balzars specimenholders (Balzars Co., Nashua, N.H.) The samples were frozen by immersionin liquid propane (−190° C.), using a guillotine-type device. The frozensamples were transferred under liquid nitrogen to the specimen state ofa Balzars, 400K freeze-fracture apparatus precooled to −150° C. Thefrozen suspension of bacteria was fractured at −120° C. using a knifecooled at the temperature of liquid nitrogen. The fracture surface wasimmediately replicated with platinum-carbon at 45′ and carbon at 90′.The replicas were floated in 3 to 4% sodium hypochlorite to bleach theorganic material, washed three times in double distilled water, andplaced on Formvar-coated freeze-fracture grids (Ted Pella Inc., Redding,Calif.). The grids were observed in a JEOL 100 CX II electron microscopeoperated at 80 kV. For each organism studied, a minimum of 50 fields wasphotographed and printed at a final magnification of ×100,000; typicalfractured cells were chosen from these fields for determination ofintramembranous particle density.

[0070] The outer membrane particle density of virulent andculture-attenuated L. alstoni serovar grippotyphosa was studied. Theresults were interesting in two respects: 1) Like other pathogenicspirochetes, this serovar of L. alstoni had a low OMP content relativeto enteric gram-negative bacteria; and 2) comparison of virulent andattenuated L. alstoni revealed a direct correlation between expressionof the 31-kDa protein (OmpL1) and integral membrane particle density.

Example 4 Cloning of the 31-KDA Protein OmpL 1

[0071] The weight of evidence from the different approaches used tostudy L. alstoni surface components led to the conclusion that the31-kDa protein was the most promising OMP candidate. The fact that theamount of the 31-kDa protein present was not correlated with virulencedoes not diminish its importance because it would be the first membranespanning spirochetal OMP to have been identified. The N-terminal aminoacid sequence of the 31-kDa protein was obtained as follows. The surfaceimmunoprecipitation procedure was used to generate a sample which wassubjected to SDS-polyacrylamide gel electrophoresis, transferred toTrans-Blot PVDF Protein Sequencing Membrane (Bio-Rad, Richmond, Calif.),and submitted to the UCLA microsequencing facility. It was possible todetermine the N-terminal 13-14 consecutive amino acids of the 31-kDaprotein. Based upon the N-terminal amino acid sequence, two degenerateoligonucleotide probes were synthesized.

[0072]L. alstoni genomic DNA was prepared by the method of Yelton, D.B., and N. W. Charon, 1948. The oligonucleotide probes were end-labeledwith gamma ³²P-ATP, and independently identified a 2.5 kb Eco RIfragment by Southern hybridization of the L. alstoni genome. An Eco RIgenomic digest was separated by agarose gel electrophoresis. Fragmentswith a size range of 2.1-2.8 kb generated by restriction endonucleases,representing 10% of the total Leptospira DNA, were removed and gelpurified. These DNA fragments were ligated into the Lambda Zap II vector(Stratagene). The oligonucleotide probes were used to independentlyscreen a library of 4800 plaques. The size of the Leptospira genome isroughly five megabases (Baril, et al. FEMS Microbiol. Lett., 71:95-100).The bacteriophage lambda library represented about 10% of the totalgenome, or 500,000 base pairs. The expected frequency of positiveplaques would be fragment size/genome fraction=2500/500,000=0.25%. Basedupon this calculation, hybridization was expected to 4800×0.25%=12plaques. Both oligonucleotide probes hybridized to the same 18 plaques.Six positive plaques were picked, replated, and reprobed in order topurify phage bearing the hybridizing DNA fragment. Purified phage wereamplified and converted to pBluescript SK-plasmid form by in vivoexcision and recircularization. All six pBluescript plasmids obtained inthis way contained the same 2.5 kb insert. A restriction map of theinsert was constructed. Southern hybridization using the oligonucleotideprobes was performed, localizing the area of hybridization to a 122 bpfragment near the Nde I site.

Example 5 Sequence Analysis for OmpL1

[0073] Restriction fragments were subcloned into pBluescript fordouble-stranded DNA sequencing using the Sequenase reaction with the T7forward and T3 reverse primers. The DNA sequence was analyzed using theDNA Strider 1.0 program. An intact open reading frame was foundbeginning 770 bases in from the upstream Eco RI site. The ompL1structural gene consists of 960 bases encoding a protein of 320 aminoacids. E. coli-like-35 (TTGCCG) and—10(TCCAAT) promoter regions arepresent upstream. An E. coli consensus ribosome-binding site (AGGAG) ispresent 6 bases upstream from the initiation codon. As expected forproteins to be exported beyond the inner membrane, the derived aminoacid sequence begins with a 24 residue signal peptide represented by theshaded area in FIG. 1. There is a leader peptidase I cleavage site, thesequence of which is typical for procaryotic exported proteins in thatthere is an alanine at the −1 position. It is also notable that theamino acid sequence does not contain a concensus Leu-X-Y-Cys leaderpeptidase II cleavage site, indicating that OmpL1 is not a lipoprotein.To test whether or not this export signal is functional in E. coli, anSsp I fragment containing the OmpL1 leader was cloned into the Sma Isite of the polylinker of the alkaline phosphatase expression vector pMGand transformed into IT41, a strain of E. coli with a temperaturesensitive leader peptidase 1 mutation (Inada, et a., J. Bacteriol.,171:585-587, 1989). The resultant OmpL1-PhoA fusion protein was exportedto the periplasm resulting in blue colonies on agar containing thealkaline phosphatase substrate XP (bromo-chloro-indolylphosphate).Cleavage of the OmpL1-PhoA fusion protein was demonstrated at thepermissive temperature of 32° C., but not at the restrictive temperatureof 37° C., confirming sensitivity of OmpL1 to E. coli leader peptidase 1cleavage. Immediately following the leader peptidase 1 cleavage site wasa sequence of 13 amino acids that agreed exactly with the first 13 aminoacids of the N-terminal amino acid sequence obtained from the nativeprotein. The 296 amino acid mature protein has a calculated molecularweight of approximately 31,000 daltons, which is similar to thatobserved experimentally. The hydrophobicity plot shows an N-terminalpeak of 0.75, corresponding to the leader peptide. There are no otherregions with a hydrophobicity score of greater than 0.65, consistentwith the idea that the ompL1 gene does not encode an inner membraneprotein. Downstream of the termination codon (TAA) is an invertedrepeat, which might function as a rho-independent transcriptionterminator, extending from nucleotides 997 to 1027.

[0074] The OmpL1 sequence contains ten stretches of amphipathicbeta-sheet structure consistent with OMP transmembrane segments. On thebasis of the primary amino acid sequence data it is possible to proposea topological model of OmpL1 which conforms to structural rules basedupon the topology of well-defined porins of gram-negative bacteria. Eachof the transmembrane segments is ten amino acids in length, and withinthese ten transmembrane segments there are no exceptions to thealternating hydrophobic amino acid rule. The amino acids in thetransmembrane segments are shown in a staggered array with thehydrophobic, membrane-facing residues on the right side of the array.Some of the transmembrane segments are reflected as peaks on thebeta-moment plot, which is designed to identify areas where hydrophobicand hydrophilic amino acids alternate and is useful in predicting OMPmembrane spanning sequences (Vogel, et al., J. Mol. Biol., 190:191-199,1986). The five surface-exposed loops of varying length contain segmentsof high surface probability. The four periplasmic loops are typical ofOMPs in that they are short, and contain amino acids, such as proline(P), glucine (G), serine (S), and asparagine (N), which are turnpromoters (Jeanteur, et al., Molec. Microbiol 5:2153-2164, 1991).

[0075] The carboxy-terminal ten amino acids of OmpL1 revealed analternating pattern of hydrophobic amino acids which is characteristicof gram-negative OMPs. Of particular interest is the fact that there isa histidine at the −3 position. Histidine is found at this position inthe carboxy-terminal transmembrane segment of class 1 (Nmp1) and class 2(Nmp2) porins of Neisseria meningitidis, as well as the pl porin ofNeisseria gonorrheae (Jeanteur, et al., Molec. Microbiol., 5:2153-2164,1991). Using the Gap Alignment program (University of Wisconsin,Genetics Computer Group), set at a gap width of 3.0, alignment of theentire OmpL1 sequence with that of Nmp1 revealed that 21% of the aminoacids were identical and another 45% were similar. Pairwise alignment ofOmpL1 with a variety of other proteins indicated that OmpL1 is morehomologous to Nmp1 than to OMPs of E. coli (such as LamB, OmpF, orPhoE), OmpL1 was more homologous to OMPs than to periplasmic proteins ofE. coli (such as AraF or beta-lactamase). The gap alignment score ofOmpL1 with these proteins is shown in brackets: Nmp1 [113.5], LamB[107.7], PhoE [104.8], AraF [95.7], and beta-lactamase [95.5]. Scores ofpairwise alignment have been used to assess relative homology of porins(Gerbl-Reiger, S., et al., J. Bacteriol, 173-2196-2205, 1991). Gapalignment of OmpL1 with Nmp1 demonstrated homology in areascorresponding to known Nmp1 transmembrane segments. Neisserial porinsshow greatest sequence conservation in their transmembrane segments (Vander Ley, P., et al., Infect. Immun. 59:2963-2971, 1991). Transmembranesegments (TMSs) #6, #7, #8, #9, #11, and #16 of Nmp1 aligned with theOmpL1 sequence in a pattern such that most of the alternatinghydrophobic residues (*) of the Nmp1 transmembrane segments wereidentical (I) or similar (:) to those in OmpL1 (FIG. 2).

Example 6 Southern Hybridization Studies

[0076] There is a strong correlation between Leptospira pathogenicityand the presence of the ompL1 gene. Southern hybridization studies wereperformed as follows. The ompL1 gene has a unique Nde I site two codonsafter the leader peptides cleavage site is encoded. There is also aunique Pvu I site near the end of the structural gene. The 834 base pairNde I-Pvu I fragment, encoding 94% of the mature protein, was labeledwith alpha ³²P-dCTP by the random priming method and used to probe EcoRI genomic digests of eleven pathogenic and four nonpathogenicLeptospira strains. FIG. 3A shows the results after washing at mediumstringency in 2×SSC at 55° C. A single copy of the gene was present inall the tested strains of pathogenic Leptospira. The gene was notpresent in nonpathogenic strains of Leptospira. FIG. 3B shows theresults after washing at high stringency in 0.1×SSC at 55° C. The mosthomologous ompL1 genes are found in other L. alstoni serovars tested.

Example 7 Cloning of the OmpL1 Gene into the pRCET Expression Vector

[0077] The pBluescript plasmid containing the ompL1 gene was digestedwith Nde I, filled in the Klenow to generate blunt ends, and thendigested with Eco RI. The resulting 1600 base pair DNA fragment encodingessentially the entire mature OmpL1 protein was isolated by agarose gelelectrophoresis, and ligated into pRSET (Invitrogen, San Diego, Calif.)digested with Sma I and Eco RI. The resulting construct, pRSET-ompL1,encodes a fusion protein containing a 41 amino acid His6 binding site atthe amino terminus of OmpL1. The six histidines allow for pH-dependentaffinity purification of the fusion protein on a nickel resin column tothe exclusion of E. coli proteins. The pRSET fusion protein is under T7promoter control. After transformation of pRSET-ompL1 into E. coli DH5α,milligram quantities of the His6-OmpL1 fusion protein were produced inthe presence of isopropyl-β-D-thiogalactoside (IPTG, Sigma). Addition ofrifampicin (Sigma) one hour after IPTG induction enhanced His6-OmpL1production relative to E. coli proteins. The bulk of the E. coli hostproteins were released after several freeze-thaw cycles in lysis buffer,while >90% of the insoluble pellet consisted of the His6-OmpL1 fusionprotein.

Example 8 Immunization of Rabbits with Purified OmpL1

[0078] The His6-OmpL1 fusion protein was separated from other insolublematerials by SDS-PAGE. The His6-OmpL1 band containing 50 micrograms ofprotein was cut out of the acrylamide gel, dessicated, ground to powder,mixed with Freund's complete adjuvant and inoculated subcutaneously andintramuscularly into a New Zealand White male rabbit. AdditionalHis6-OmpL1 fusion protein was solubilized in 6M guanidine and purifiedover the nickel resin column and dialyzed in 10 mM Tris, pH 8.0. Thesecondary immunization was given six weeks after the primaryimmunization using roughly 50 micrograms of purified His6-OmpL1 fusionprotein in Freund's incomplete adjuvant. The rabbit was bled two weeksafter the secondary immunization. The post-boost antiserum reacted onlywith the 31-kDa antigen on immunoblots of whole L. alstoni separated bySDS-PAGE. Immunoblots of L. Alstoni fractioned with TX-114 revealedreactivity with the 31-kDa OmpL1 antigen in the whole organism anddetergent phase, but not the aqueous phase or insoluble pellet.

Example 9 Surface Localization with Immunoelectron Microscopy

[0079] Having obtained a highly specific immunological reagent forlocalization studies, preliminary immunoelectron microscopy experimentswere conducted. A 20 μl suspension of 10⁷ L. alstoni was added to 0.5 mlof heat-inactivated anti-OmpL1 antiserum or preimmune serum from thesame rabbit and incubated for one hour with mixing. The bacteria werefixed for 30 minutes by addition of 250 μl of 0.75% glutaraldehyde in100 mM cacodylate buffer, pH 7.0. The bacteria were washed, applied toelectron microscopy grids, and probed with protein G-colloidal gold (10nm particles). A low level of specific binding to the outer membrane ofL. alstoni was observed. With preimmunization serum, one particle pertwenty organisms was observed, whereas with anti-OmpL1 antiserum eightparticles per twenty organisms was observed. A low level of binding wasanticipated because of the paucity of leptospiral OMPs.

Example 10 Expression of OmpL1 with the pTrc 99A Expression Vector

[0080] The His6 fusion protein is well suited for purification, but isnot appropriate for immunoblotting studies because of the potential forbackground reactivity to the 41 additional amino acids containing theHis6 binding site. Preimmune sera from one of the rabbits reacted withthe His6-OmpL1 fusion protein, but not with native OmpL1. A BgI II-HindII fragment was isolated from the pRCET-ompL1 vector by gelelectrophoresis and cloned into the pTrc99A expression vector(Pharmacia) which had been reading frame adjusted with a 10-mer Nco Ilinker. The pTtrc99A-ompL1 construct, transformed into E. coli DH5αexpresses the entire mature OmpL1 protein, plus a start methionine andonly five additional amino acids supplied by the vector. E. coli DH5αcontaining the original pTrc99A vector served as a negative control.Bacterial proteins were separated by SDS-PAGE and transferred tonitrocellulose, and probed with antisera from rabbits immunized with avariety of pathogenic Leptospira strains (antisera supplied by Dr.Arnold Kaufmann, Centers for Disease Control, Atlanta). Reactivity toOmpL1 was demonstrated with antisera to L. interrogans, serovarsicterohaemorrhagiae, pomona, and bratislava, L. alstoni, serovarsgrippotyphosa and fortbragg, L. santarosai, serovars bakeri andcanalzonae, and L. weilii, serovar celledoni. This indicates that OmpL1is not only expressed, but also antigenically conserved among pathogenicLeptospira, a feature that would make it an excellent vaccine candidate.

[0081] The foregoing is meant to illustrate, but not to limit, the scopeof the invention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

Summary of Sequences

[0082] Sequence I.D. No. 1 is the nucleic acid sequence of ompL1.

[0083] Sequence l.D. No. 2 is the deduced amino acid sequence of OmpL1.

1 7 963 base pairs nucleic acid single linear DNA (genomic) OMP L1 CDS1..963 1 ATG ATC CGT AAC ATA AGT AAG GCA TTG CTC ATT TTA GCC GTA GCA CTA48 Met Ile Arg Asn Ile Ser Lys Ala Leu Leu Ile Leu Ala Val Ala Leu 1 510 15 TCT TCG GCT GCA AGC CTA AGT GCA AAA ACA TAT GCA ATT GTA GGA TTT 96Ser Ser Ala Ala Ser Leu Ser Ala Lys Thr Tyr Ala Ile Val Gly Phe 20 25 30GGG TTA CAG TTA GAC CTG GGA CAA TTA GGA GGA ACC ATC ACT AAA GAC 144 GlyLeu Gln Leu Asp Leu Gly Gln Leu Gly Gly Thr Ile Thr Lys Asp 35 40 45 GGTTTG GAC GCT GCG AGT TAT TAT GGT CCA GTC CGA TCA ACA GAT ACT 192 Gly LeuAsp Ala Ala Ser Tyr Tyr Gly Pro Val Arg Ser Thr Asp Thr 50 55 60 TGT ACAGTA GGT CCA AAC GAT CCT ACT TGT GTA CAA AAT CCA GGA AAA 240 Cys Thr ValGly Pro Asn Asp Pro Thr Cys Val Gln Asn Pro Gly Lys 65 70 75 80 CCT GCAGGT GAA GGA AAT TAT CTA GGA GTT GCT CCT AGA AAA GCG ATT 288 Pro Ala GlyGlu Gly Asn Tyr Leu Gly Val Ala Pro Arg Lys Ala Ile 85 90 95 CCC GCT GAAAAT AAA TTG ATT ACC CTC GAT AGA ACT ACT GGC GGT TTG 336 Pro Ala Glu AsnLys Leu Ile Thr Leu Asp Arg Thr Thr Gly Gly Leu 100 105 110 ATC AAT GCGAGA AGC ACC AAA GGA GCC ATG GTC GGA GGA AAT TTG ATG 384 Ile Asn Ala ArgSer Thr Lys Gly Ala Met Val Gly Gly Asn Leu Met 115 120 125 GTA GGT TACGAA TCC GAC TTT GGT AAA TAT TTT TTC TGG AGA GTT GCT 432 Val Gly Tyr GluSer Asp Phe Gly Lys Tyr Phe Phe Trp Arg Val Ala 130 135 140 GCA GAA TATACT CAA AAA ATT TCC GGT GGT ATT ACA AAA GCG GAC ATC 480 Ala Glu Tyr ThrGln Lys Ile Ser Gly Gly Ile Thr Lys Ala Asp Ile 145 150 155 160 GCT GGTTAT AGT ATT GTA GAC ATG ACC TGG GGA TTT AGT TCT ATC GTC 528 Ala Gly TyrSer Ile Val Asp Met Thr Trp Gly Phe Ser Ser Ile Val 165 170 175 ATT CCTGCA ACT GTT GGT ATT AAA TTG AAT GTT ACT GAA GAC GCT GCT 576 Ile Pro AlaThr Val Gly Ile Lys Leu Asn Val Thr Glu Asp Ala Ala 180 185 190 GTG TATATG GGA GCC GGT CTG AAC TAC TTT AAC GGC TGG TGG AGT TTA 624 Val Tyr MetGly Ala Gly Leu Asn Tyr Phe Asn Gly Trp Trp Ser Leu 195 200 205 AAC GGATCC AAT AAC CTC AAA GGA GGT CAT GAC ATT TTA GCC GCA GCG 672 Asn Gly SerAsn Asn Leu Lys Gly Gly His Asp Ile Leu Ala Ala Ala 210 215 220 GGA GCAGGA AGT GTT GCA AAC TTA ATC GCA GAC GGA ACG GAT CCA ATC 720 Gly Ala GlySer Val Ala Asn Leu Ile Ala Asp Gly Thr Asp Pro Ile 225 230 235 240 ACTACT CGT GAG CAC GTT CGT TTT AGA ACT TCT GGA ATT GCT CCT AAC 768 Thr ThrArg Glu His Val Arg Phe Arg Thr Ser Gly Ile Ala Pro Asn 245 250 255 TTTTTA ATT GGA ACC CAA GCC AGA GTA ACC GAC AAA GGA CAC GTT TTT 816 Phe LeuIle Gly Thr Gln Ala Arg Val Thr Asp Lys Gly His Val Phe 260 265 270 CTTGAA TTA GAA ACG ATC ATG TCT GCT GCG TAT GCA GTT GGT AAA ACT 864 Leu GluLeu Glu Thr Ile Met Ser Ala Ala Tyr Ala Val Gly Lys Thr 275 280 285 CAATCT GCT GGA GGA GCC ACG AAT CTT TCT CCT TTT CCA GCG TAT CCG 912 Gln SerAla Gly Gly Ala Thr Asn Leu Ser Pro Phe Pro Ala Tyr Pro 290 295 300 ATCGTT GTC GGT GGG CAA ATC TAC AGA TTC GGT TAT AAA CAC GAA CTC 960 Ile ValVal Gly Gly Gln Ile Tyr Arg Phe Gly Tyr Lys His Glu Leu 305 310 315 320TAA 963 320 amino acids amino acid linear protein 2 Met Ile Arg Asn IleSer Lys Ala Leu Leu Ile Leu Ala Val Ala Leu 1 5 10 15 Ser Ser Ala AlaSer Leu Ser Ala Lys Thr Tyr Ala Ile Val Gly Phe 20 25 30 Gly Leu Gln LeuAsp Leu Gly Gln Leu Gly Gly Thr Ile Thr Lys Asp 35 40 45 Gly Leu Asp AlaAla Ser Tyr Tyr Gly Pro Val Arg Ser Thr Asp Thr 50 55 60 Cys Thr Val GlyPro Asn Asp Pro Thr Cys Val Gln Asn Pro Gly Lys 65 70 75 80 Pro Ala GlyGlu Gly Asn Tyr Leu Gly Val Ala Pro Arg Lys Ala Ile 85 90 95 Pro Ala GluAsn Lys Leu Ile Thr Leu Asp Arg Thr Thr Gly Gly Leu 100 105 110 Ile AsnAla Arg Ser Thr Lys Gly Ala Met Val Gly Gly Asn Leu Met 115 120 125 ValGly Tyr Glu Ser Asp Phe Gly Lys Tyr Phe Phe Trp Arg Val Ala 130 135 140Ala Glu Tyr Thr Gln Lys Ile Ser Gly Gly Ile Thr Lys Ala Asp Ile 145 150155 160 Ala Gly Tyr Ser Ile Val Asp Met Thr Trp Gly Phe Ser Ser Ile Val165 170 175 Ile Pro Ala Thr Val Gly Ile Lys Leu Asn Val Thr Glu Asp AlaAla 180 185 190 Val Tyr Met Gly Ala Gly Leu Asn Tyr Phe Asn Gly Trp TrpSer Leu 195 200 205 Asn Gly Ser Asn Asn Leu Lys Gly Gly His Asp Ile LeuAla Ala Ala 210 215 220 Gly Ala Gly Ser Val Ala Asn Leu Ile Ala Asp GlyThr Asp Pro Ile 225 230 235 240 Thr Thr Arg Glu His Val Arg Phe Arg ThrSer Gly Ile Ala Pro Asn 245 250 255 Phe Leu Ile Gly Thr Gln Ala Arg ValThr Asp Lys Gly His Val Phe 260 265 270 Leu Glu Leu Glu Thr Ile Met SerAla Ala Tyr Ala Val Gly Lys Thr 275 280 285 Gln Ser Ala Gly Gly Ala ThrAsn Leu Ser Pro Phe Pro Ala Tyr Pro 290 295 300 Ile Val Val Gly Gly GlnIle Tyr Arg Phe Gly Tyr Lys His Glu Leu 305 310 315 320 14 amino acidsamino acid single linear peptide Peptide 1..14 3 Lys Thr Tyr Ala Ile ValGly Phe Gly Leu Gln Leu Asp Asn 1 5 10 20 base pairs nucleic acid singlelinear DNA (genomic) CDS 1..20 4 AARACNTAYG CNATHGTNGG 20 20 base pairsnucleic acid single linear DNA (genomic) CDS 1..20 5 TTYGGNYTDCARYTDGAYAA 20 60 amino acids amino acid single linear peptide Nmp1Peptide 1..60 6 Val Ser Val Arg Tyr Asp Ser Phe Ser Gly Phe Ser Gly SerVal Gln 1 5 10 15 Phe Tyr Tyr Ala Gly Leu Asn Tyr Phe Ala Gly Asn TyrAla Phe Lys 20 25 30 Tyr Ala Lys Gly Thr Asp Pro Leu Asn Leu Ala Leu AlaAla Gln Leu 35 40 45 Asp Leu Ala Ala Ser Val Gly Leu Arg His Lys Phe 5055 60 60 amino acids amino acid single linear peptide PepL Peptide 1..607 Leu Met Val Gly Tyr Glu Ser Tyr Phe Phe Trp Arg Val Ala Ala Glu 1 5 1015 Tyr Met Gly Ala Gly Leu Asn Tyr Phe Asn Gly Trp Trp Ser Leu Asn 20 2530 Gly Ala Asp Gly Thr Asp Pro Ile Asn Phe Leu Ile Gly Thr Gln Ala 35 4045 Arg Val Ile Tyr Arg Phe Gly Tyr Lys His Glu Leu 50 55 60

1. An isolated polypeptide comprising the amino acid sequence of OmpL1.2. The polypeptide of claim 1, wherein the polypeptide has a molecularweight of about 31 kD as determined by SDS-PAGE.
 3. The polypeptide ofclaim 2, wherein the polypeptide has essentially the amino acid sequenceof FIG.
 1. 4. The polypeptide of claim 1, wherein the OmpL1 is fromLeptospira alstoni.
 5. The polypeptide of claim 4, wherein the OmpL1 isfrom a serovar of Leptaspira alstoni selected the group consisting ofgrippotyphosa and fortbragg.
 6. The polypeptide of claim 1, wherein theOmpL1 is from Leptospira interrogans.
 7. The polypeptide of claim 6,wherein the OmpL1 is from a serovar of Leptospira interrogans selectedfrom the group consisting of icterohaemorrhagiae, pomona and bratislava.8. An isolated polynucleotide sequence which encodes the polypeptide ofclaim
 1. 9. The polynucleotide sequence of claim 8, wherein thepolynucleotide is DNA.
 10. The polynucleotide sequence of claim 8,wherein the polynucleotide is RNA.
 11. A recombinant expression vectorcontaining the polynucleotide of claim
 8. 12. The expression vector ofclaim 11, wherein the vector is a plasmid.
 13. The vector of claim 11,wherein the polynucleotide sequence is from L. alstoni.
 14. A host celltransformed with the expression vector of claim
 11. 15. The host cell ofclaim 14, wherein the cell is a prokaryote.
 16. The prokaryote of claim15, which is E. coli.
 17. The host cell of claim 14, wherein the cell isa eukaryote.
 18. A method of producing OmpL1 polypeptide whichcomprises: a. transforming a host with the polynucleotide of claim 8;and b. expressing the polynucleotide in the host.
 19. The method ofclaim 18, which further comprises isolating the OmpL1 polypeptide. 20.The method of claim 18, wherein the host is a prokaryote.
 21. Apharmaceutical composition useful for inducing an immune response topathogenic Leptospira in an animal comprising an immunogenicallyeffective amount of OmpL1 in a pharmaceutically acceptable carrier. 22.The pharmaceutical composition of claim 21, wherein the pharmaceuticallyacceptable carrier contains an adjuvant.
 23. A method of inducing animmune response to pathogenic Leptospira in an animal comprisingimmunizing the animal with the composition of claim
 21. 24. Apharmaceutical composition useful for inducing an immune response topathogenic Leptospira in an animal comprising an immunogenicallyeffective amount of antibody which binds OmpL1 in a pharmaceuticallyacceptable carrier.
 25. An antibody which binds to OmpL1.
 26. Theantibody of claim 25, wherein the antibody is polyclonal.
 27. Theantibody of claim 25, wherein the antibody is monoclonal.
 28. A methodof detecting a pathogenic Leptospira in a sample comprising contacting apathogen-specific cell component in the sample with a reagent whichbinds to the pathogen-specific cell component.
 29. The method of claim28, wherein the pathogen-specific cell component is nucleic acid whichencodes OmpL1 polypeptide.
 30. The method of claim 29, wherein thenucleic acid is DNA.
 31. The method of claim 28, wherein the nucleicacid is RNA.
 32. The method of claim 28, wherein the pathogen specificcell component is OmpL1 polypeptide.
 33. The method of claim 28, whereinthe reagent is a probe.
 34. The method of claim 33, wherein the probe isnucleic acid.
 35. The method of claim 33, wherein the probe is anantibody.
 36. The method of claim 35, wherein the antibody ispolyclonal.
 37. The method of claim 35, wherein the antibody ismonoclonal.
 38. The method of claim 33, wherein the probe is detectablylabeled.
 39. The method of claim 38, wherein the label is selected fromthe group consisting of a radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate, oran enzyme.
 40. The method of claim 28, wherein the sample is from ananimal selected from the group consisting of human, swine and cattle.41. A method for detecting antibody to OmpL1 polypeptide in a samplewhich comprises contacting the sample with OmpL1 polypeptide underconditions which allow the antibody to bind to OmpL1 polypeptide anddetecting the binding of the antibody to the OmpL1 polypeptide.
 42. Themethod of claim 41, wherein the OmpL1 polypeptide is detectablylabelled.
 43. A kit useful for the detection of OmpL1, the kitcomprising carrier means being compartmentalized to receive in closeconfinement therein one or more containers comprising containercontaining a OmpL1 binding reagent.
 44. The kit of claim 43, wherein thereagent is an antibody.
 45. The kit of claim 44, wherein the antibody ishuman.
 46. The kit of claim 44, wherein the antibody is monoclonal. 47.A kit useful for the detection of OmpL1 the kit comprising carrier meansbeing compartmentalized to receive in close confinement therein one ormore containers comprising container containing a OmpL1 polynucleotidebinding reagent.
 48. The kit of claim 47, wherein the binding reagent isnucleic acid.
 49. A kit useful for the detection of antibody to OmpL1polypeptide, the kit comprising carrier means being compartmentalized toreceive in close confinement therein one or more containers comprisingcontainer containing OmpL1 polypeptide.